Measles virus vaccine expressing sars-cov-2 protein(s)

ABSTRACT

A recombinant measles viral vector comprising a nucleic acid sequence encoding a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein is provided. Polypeptides comprising the SARS-CoV-2 spike glycoprotein also are provided, as well as related nucleic acids, vectors, and compositions. The polypeptides, nucleic acids, vectors, and compositions can be used in methods of preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infectious clinical condition caused by coronavirus and methods for inducing an immune response against a coronavirus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 63/039,275, filed Jun. 15, 2020, and U.S. ProvisionalPatent Application No. 63/071,479, filed Aug. 28, 2020, each of which isincorporated by reference herein in its entirety.

SEQUENCE LISTING

Incorporated by reference in its entirety herein is a nucleotide/aminoacid sequence listing submitted concurrently herewith and identified asfollows: One 337,215 Byte ASCII (Text) file named “750663.TXT,” createdon Aug. 31, 2020.

BACKGROUND OF THE INVENTION

In December 2019, a novel coronavirus (Severe Acute Respiratory SyndromeCoronavirus 2 or SARS-CoV-2) belonging to the betacoronavirus familyemerged. All human betacoronaviruses are unique from one another,however, they do share a certain degree of genetic and structuralhomology. SARS-CoV-2 genome sequence homology with SARS-CoV and MERS-CoVis 77% and 50%, respectively.

In contrast to the relatively smaller outbreaks of SARS-CoV in 2002 andMERS-CoV in 2012, SARS-CoV-2 is exhibiting an unprecedented scale ofinfection, resulting in a global pandemic declaration of CoronavirusInfectious Disease (COVID-19) by the World Health Organization (WHO).COVID-19 has a high infection rate and long incubation period. Similarto influenza, COVID-19 has the potential to become a seasonal disease.

Therefore, there is a desire for a COVID-19 vaccine.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a recombinant measles viralvector (rMV) (e.g., Edmonston Zagreb (EZ) MV) comprising a nucleic acidsequence encoding a Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2) spike (S) glycoprotein.

An embodiment of the invention provides a pharmaceutical compositioncomprising the recombinant measles viral vector, as well as methods forpreventing, inhibiting, reducing, eliminating, protecting, or delayingthe onset of an infection or an infectious clinical condition caused bycoronavirus in a subject and methods for inducing an immune responseagainst a coronavirus in a subject.

An embodiment of the invention also provides a polypeptide comprising anamino acid sequence selected from the group consisting of the amino acidsequences of SEQ ID NO: 8 (S6), SEQ ID NO: 9 (S-CO), SEQ ID NO: 10(S-CO-AA), SEQ ID NO: 11 (S), SEQ ID NO: 12 (S-CO-AA-PP), SEQ ID NO: 13(S-CO-AA-fneg-PP), and SEQ ID NO: 14 (S-CO-AA-fneg), as well as relatednucleic acids, recombinant vectors, compositions, and methods.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1C demonstrate the generation of rMV^(EZ) viruses expressingSARS-CoV-2 spike glycoprotein. FIG. 1A is a schematic representation ofparental virus rMV^(EZ)EGFP(3), the cDNA clone of which was used togenerate cDNA plasmids for viruses rMV^(EZ)SARS-CoV-2-S3,rMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S-CO andrMV^(EZ)SARS-CoV-2-S-COAA. Amino acid differences in the spike betweenthe four viruses are shown, wherein underlined residues differ fromwild-type spike sequence. Residues K and H in the cytoplasmic tailregion were altered to A and A to disrupt a endoplasmic reticulum (ER)retention signal (KxHxx) sequence in viruses rMV^(EZ)SARS-CoV-2-S3,rMV^(EZ)SARS-CoV-2-S6 and rMV^(EZ)SARS-CoV-2-S-COAA. RBD; receptorbinding domain, TM; transmembrane domain. FIG. 1B is a phase image ofVero cell monolayer infected with rMV^(EZ)SARS-CoV-2-S6 (shown as arepresentative image of a primary rescue). FIG. 1C are images of animmunoplaque assay of Vero cell monolayers infected with a 10-folddilution of rescued rMV^(EZ)SARS-CoV-2-S6. Plaques were stained usinganti-SARS-CoV-2-S antibody. Dilutions 10⁻⁶ and 10⁻⁷ are shown asrepresentatives.

FIG. 2 demonstrates that growth kinetics of rMV^(EZ)SARS-CoV-2-S3,rMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S-CO, andrMV^(EZ)SARS-CoV-2-S-COAA compared to rMV^(EZ)EGFP(3). Vero cells wereinfected at a multiplicity of infections (MOI) of 0.05. Samples wereharvested at the indicated time points and titrated on Vero cells. Errorbars indicate standard deviation (n=3).

FIGS. 3A-3B demonstrate an experimental set-up for mice immunized withrecombinant viruses. FIG. 3A is a schematic describing the experiment.Groups of 5 mice were infected with either rMV^(EZ)SARS-CoV-2-S3,rMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S-CO,rMV^(EZ)SARS-CoV-2-S-COAA, or rMV^(EZ)EGFP(3). Serum was collected ondays 21 and 42, and splenocytes were collected on day 42. FIG. 3B istable showing neutralization of SARS-CoV-2 using the harvested miceserum, indicating that SARS-CoV-2 neutralizing antibodies were producedin mice vaccinated with rMV^(EZ)SARS-CoV-2-S-CO andrMV^(EZ)SARS-CoV-2-S-COAA viruses.

FIG. 4 is a vector map of rMV^(EZ)SARS-CoV-2-S6.

FIG. 5 is a vector map of rMV^(EZ)SARS-CoV-2-S-CO.

FIG. 6 is a vector map of rMV^(EZ)SARS-CoV-2-S-COAA.

FIG. 7 is a vector map of rMV^(EZ)SARS-CoV-2-S.

FIG. 8 is a vector map of rMV^(EZ)SARS-CoV-2-S-COAA-PP.

FIG. 9 is a vector map of rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP.

FIG. 10 is a vector map of and rMV^(EZ)SARS-CoV-2-S-COAA-fneg.

FIGS. 11A-B demonstrates an experimental set-up for non-human primatesvaccinated (prime and/or boost) with any of the candidates. FIG. 11A isa schematic describing the experiment. Groups of 2 or 3 African greenmonkeys (AGMs) were vaccinated with rMV^(EZ)SARS-CoV-2-S-CO or rMV^(EZ)and then challenged with SARS-CoV-2. FIG. 11B is a table withneutralization titers of serum against SARS-CoV-2, which were determinedas described in Klimstra et al., 2020: PMID: 32821033, and MV asdescribed in de Swart et al., 2017: PMID: 29263877. The results supportthat primates produce antibodies which neutralize both SARS-CoV-2 andmeasles virus following vaccination.

FIG. 12 is a graph demonstrating the secretion of IFN-γ in spleens frommice immunized with recombinant measles virus expressing SARS-CoV-2codon-optimized spike protein. Splenocytes prepared from rMV^(EZ),rMV^(EZ)SARS-CoV-2-CO, rMV^(EZ)SARS-CoV-2-S6, andrMV^(EZ)SARS-CoV-2-COAA immunized mice were used in an ELISPOT assay todetect the secretion of proinflammatory cytokine IFN-γ. Mice splenocyteswere re-stimulated for 24 hours with four pools of synthetic peptides(S1, S2, S3 and S4) designed to span the entire SARS-CoV-2 spikeprotein. Unstimulated splenocytes (medium) served as negative controlsand splenocytes treated with PMA/ionomycin confirmed splenocytere-stimulation. Dots represent individual animals (n=5) for eachvaccinated group. The number of cells expressing IFN-γ afterre-stimulation are represented as 1×10⁵ cells.

FIGS. 13A and 13B demonstrate the T cell responses in immunized mice.Flow cytometry was used to determine the proportion of CD4⁺/CD44⁺ andCD8⁺/CD44⁺ T cells in the spleens of mice immunized with rMV^(EZ),rMV^(EZ)SARS-CoV-2-CO, rMV^(EZ)SARS-CoV-2-S6, andrMV^(EZ)SARS-CoV-2-COAA. To determine the optimal re-stimulation periodsplenocytes from two mice from each immunized group were firstre-stimulated for 6 hours (FIG. 13A) while the remaining three micespleens were re-stimulated for 12-hours (FIG. 13B). Cells werere-stimulated with spike specific peptide pools S1, S2, S3 and S4, orleft unstimulated (negative control; medium). Re-stimulation wasconfirmed with a cell activation cocktail containing PMA/ionomycin andbrefaldin-A. Intracellular cytokine staining for IFN-γ and IL-13 werethen carried out to determine Th1 and Th2 responses, respectively. Dotsrepresent individual animals.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a recombinant measles viralvector that encodes one or more (e.g., two, three, four, or five)SARS-CoV-2 spike glycoproteins, wherein the SARS-CoV-2 spikeglycoproteins may be the same or different. The recombinant measlesviral vector or a composition thereof can be administered to a subjectto prevent, inhibit, reduce, eliminate, protect, or delay the onset ofan infection or an infectious clinical condition caused by coronavirus(e.g., SARS-CoV-2) in a subject. The recombinant measles viral vector ora composition thereof also can be administered to a subject to induce animmune response against a coronavirus (e.g., SARS-CoV-2) in a subject.

The recombinant measles viral vector can be any suitable recombinantmeasles viral vector. For example, the measles viral vector can be anEdmonston wild-type virus or vaccine strains of the Edmonston lineage,such as the AIK-C, Moraten, Rubeovax, Schwarz, or Zagreb strains(Bankamp et al., J. Infect. Dis., 204 Suppl. 1: 5533-5548 (2011)).Alternatively, the measles viral vector can be selected from the groupconsisting of CAM-70, Changchun-47, Leningrad-4, Shanghai-191 (Bankampet al., J. Infect. Dis., 204 Suppl. 1: 5533-5548 (2011)), Leningrad-16,Moscow-5 (Sinitsyna et al., Res. Virol., 141(5): 517-31 (1990)), 9301B(Takeda et al., J Virol., 72(11): 8690-8696 (1998)), ATTENUVAX®, andthose described in Schneider-Schaulies et al., PNAS, 92(2): 3943-7(1995).

Measles viruses and recombinant measles viral vectors are described inWO 98/13501, which provides the sequence of a DNA copy of the positivestrand (antigenomic) message sense RNA of various wild-type of vaccinemeasles strains, including Edmonston wild-type strain, Moraten strain,and Schwarz strain, and WO 97/06270, which discloses the production ofrecombinant measles vectors.

In one embodiment, the measles viral vector is an Edmonston-Zagreb (EZ)measles viral vector. Particular recombinant measles viral vectors ofthe Edmonston-Zagreb (EZ) strain include the following:rMV^(EZ)SARS-CoV-2-S6 (FIG. 4), rMV^(EZ)SARS-CoV-2-S-CO (FIG. 5),rMV^(EZ)SARS-CoV-2-S-COAA (FIG. 6), rMV^(EZ)SARS-CoV-2-S(FIG. 7),rMV^(EZ)SARS-CoV-2-S-COAA-PP (FIG. 8), rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP(FIG. 9), and rMV^(EZ)SARS-CoV-2-S-COAA-fneg (FIG. 10).

The recombinant measles vector comprises a nucleic acid encoding theSARS-CoV-2 spike glycoprotein, wherein the nucleic acid sequenceencoding the SARS-CoV-2 spike glycoprotein can be any suitable nucleicacid sequence.

In one embodiment, the nucleic acid sequence encoding the SARS-CoV-2spike glycoprotein is codon optimized. Without being bound to aparticular theory or mechanism, it is believed that codon optimizationof the nucleotide sequence increases the translation efficiency of themRNA transcripts. Codon optimization of the nucleotide sequence mayinvolve substituting a native codon for another codon that encodes thesame amino acid, but can be translated by ribosomes using tRNAs that aremore readily available within a cell, thus increasing translationefficiency and overall protein production. Optimization of thenucleotide sequence may also reduce secondary mRNA structures that wouldinterfere with translation, thus increasing translation efficiency.rMV^(EZ)SARS-CoV-2-S-CO, rMV^(EZ)SARS-CoV-2-S-COAA,rMV^(EZ)SARS-CoV-2-S-COAA-PP, rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP, andrMV^(EZ)SARS-CoV-2-S-COAA-fneg each contain codon optimized SARS-CoV-2spike glycoprotein sequences. Techniques for codon optimization areknown in the art.

The nucleic acid sequence encoding the SARS-CoV-2 spike glycoprotein cancontain at least one modification that disrupts the endoplasmicreticulum (ER) retention sequence of the SARS-CoV-2 spike glycoprotein.For example, the modification can result in an ER retention sequence ofthe SARS-CoV-2 spike glycoprotein containing AxAxx rather than KxHxx inthe cytoplasmic tail (as described in Case et al., bioRxiv (2020).doi:10.1101/2020.05.18.102038 and Example 2). rMV^(EZ)SARS-CoV2-S6,rMV^(EZ)SARS-CoV-2-S, rMV^(EZ)SARS-CoV-2-S-COAA,rMV^(EZ)SARS-CoV-2-S-COAA-PP, rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP, andrMV^(EZ)SARS-CoV-2-S-COAA-fneg each contain modifications to ablate theER retention signal. In particular, the amino acid sequences of theSARS-CoV-2 spike glycoproteins encoded by rMV^(EZ)SARS-CoV2-S6,rMV^(EZ)SARS-CoV-2-S, rMV^(EZ)SARS-CoV-2-S-COAA, rMV^(EZ)SARS-CoV2-S,rMV^(EZ)SARS-CoV-2-S-COAA-PP, rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP, andrMV^(EZ)SARS-CoV-2-S-COAA-fneg each contain two alanine substitutions atresidues 1269 and 1271 of SEQ ID NOs: 8 and 10-14, respectively.

The nucleic acid encoding the SARS-CoV-2 spike glycoprotein can containat least one modification that locks in the prefusion conformation. Thespike (S) glycoprotein is a trimeric class I fusion protein that existsin a metastable prefusion conformation that undergoes a substantialstructural rearrangement to fuse the viral membrane with the host cellmembrane (Wrapp et al., Science, 13: 1260-1263 (2020)). This process istriggered when the 51 subunit binds to a host cell receptor. Receptorbinding destabilizes the prefusion trimer, resulting in shedding of the51 subunit and transition of the S2 subunit to a stable postfusionconformation. To engage a host cell receptor, the receptor-bindingdomain (RBD) of 51 undergoes hinge-like conformational movements thattransiently hide or expose the determinants of receptor binding. Theamino acid sequences of the SARS-CoV-2 spike glycoproteins encoded byrMV^(EZ)SARS-CoV-2-S-COAA-PP and rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP eachcontain two proline modifications (PP) at residues 986 and 987 of SEQ IDNOs: 9 and 10, respectively, which lock in the prefusion conformation.

The nucleic acid encoding the SARS-CoV-2 spike glycoprotein can containat least one modification that ablates the furin cleavage signal, suchas by modifying the furin cleavage site so that furin no longer cleavesthe sequence. The furin cleavage site can be any polypeptide sitecleavable by furin. The minimal cleavage site typically is, in thesingle letter code for amino acid residues, R-X-X-R, with cleavageoccurring after the second “R” (Duckert et al., Protein Engineering,Design & Selection, 17(1):107-112 (2004); and WO 2009/032954). Whetheror not any particular sequence is cleavable by furin can be determinedby methods known in the art. For example, whether or not a sequence iscleavable by furin can be tested by incubating the sequence with furin.

rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP and rMV^(EZ)SARS-CoV-2-S-COAA-fnegeach contain modifications to ablate the furin cleavage signal. Inparticular, the amino acid sequences of the SARS-CoV-2 spikeglycoproteins encoded by rMV^(EZ)SARS-CoV2-S-COAA-fneg-PP andrMV^(EZ)SARS-CoV-2-S-COAA-fneg each contain four amino acid changes(ASVG; SEQ ID NO: 23) at residues 682-685 of SEQ ID NOs: 13 and 14,respectively, that ablate the furin cleavage signal.

In one embodiment, the nucleic acid encoding the SARS-CoV-2 spikeglycoprotein encodes an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8 (S6 spike glycoprotein), SEQ ID NO: 9 (S-COspike glycoprotein), SEQ ID NO: 10 (S-CO-AA spike glycoprotein), SEQ IDNO: 11 (S spike glycoprotein), SEQ ID NO: 12 (S-CO-AA-PP spikeglycoprotein), SEQ ID NO: 13 (S-CO-AA-fneg-PP spike glycoprotein), andSEQ ID NO: 14 (S-CO-AA-fneg spike glycoprotein).

In another embodiment, the nucleic acid encoding the SARS-CoV-2 spikeglycoprotein is selected from the group consisting of SEQ ID NO: 1 (S6),SEQ ID NO: 2 (S-00), SEQ ID NO: 3 (S-CO-AA), SEQ ID NO: 4 (S), SEQ IDNO: 5 (S-CO-AA-PP), SEQ ID NO: 6 (S-CO-AA-fneg-PP), and SEQ ID NO: 7(S-CO-AA-fneg).

The recombinant measles viral vector comprising a nucleic acid sequenceencoding a SARS-CoV-2 spike glycoprotein can have any suitable nucleicacid sequence. For example, the recombinant measles viral vector cancomprise, consist essentially of, or consist of a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 15(rMV^(EZ)SARS-CoV-2-S6), SEQ ID NO: 16 (rMV^(EZ)SARS-CoV-2-S-CO), SEQ IDNO: 17 (rMV^(EZ)SARS-CoV-2-S-COAA), SEQ ID NO: 18(rMV^(EZ)SARS-CoV-2-S), SEQ ID NO: 19 (rMV^(EZ)SARS-CoV-2-S-COAA-PP),SEQ ID NO: 20 (rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP), and SEQ ID NO: 21(rMV^(EZ)SARS-CoV-2-S-COAA-fneg). The vector sequences can comprise oneor more nucleic acid sequences encoding the N, P, S, M, F, H, and Lgenes as described in GenBank Accession Nos. AY486083.1 and AY486084.1.

An embodiment of the invention also provides a polypeptide comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 8 (S6), SEQ ID NO: 9(S-CO), SEQ ID NO: 10 (S-CO-AA), SEQ ID NO: 11 (S), SEQ ID NO: 12(S-CO-AA-PP), SEQ ID NO: 13 (S-CO-AA-fneg-PP), and SEQ ID NO: 14(S-CO-AA-fneg), corresponding a SARS-CoV-2 spike glycoprotein.

The polypeptide can be prepared by any of a number of conventionaltechniques. In this respect, the polypeptide sequence can be synthetic,recombinant, isolated, and/or purified.

The polypeptide can be isolated or purified from a recombinant source.For instance, a DNA fragment encoding a desired polypeptide can besubcloned into an appropriate vector using well-known molecular genetictechniques. The fragment can be transcribed and the polypeptidesubsequently translated in vitro. Commercially available kits also canbe employed. The polymerase chain reaction optionally can be employed inthe manipulation of nucleic acids.

The polypeptide also can be synthesized using an automated peptidesynthesizer in accordance with methods known in the art. Alternately,the polypeptide can be synthesized using standard peptide synthesizingtechniques well-known to those of skill in the art. In particular, thepolypeptide can be synthesized using the procedure of solid-phasesynthesis. If desired, this can be done using an automated peptidesynthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups andseparation of the polypeptide from the resin can be accomplished by, forexample, acid treatment at reduced temperature. The protein-containingmixture then can be extracted, for instance, with diethyl ether, toremove non-peptidic organic compounds, and the synthesized polypeptidecan be extracted from the resin powder (e.g., with about 25% w/v aceticacid). Following the synthesis of the polypeptide, further purification(e.g., using HPLC) optionally can be performed in order to eliminate anyincomplete proteins, polypeptides, peptides or free amino acids. Aminoacid and/or HPLC analysis can be performed on the synthesizedpolypeptide to validate its identity. For other applications accordingto the invention, it may be preferable to produce the polypeptide aspart of a larger fusion protein, either by chemical conjugation orthrough genetic means, such as are known to those skilled in the art. Inthis regard, an embodiment of the invention also provides a fusionprotein comprising the polypeptide and one or more other protein(s)having any desired properties or functions, such as to facilitateisolation, purification, analysis, or stability of the fusion protein.

An embodiment of the invention also provides a nucleic acid encoding thepolypeptide. In one embodiment, the nucleic acid comprises, consistsessentially of, or consists of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1 (S6), SEQ ID NO: 2 (S-CO), SEQ ID NO: 3(S-CO-AA), SEQ ID NO: 4 (SARS-CoV2-S), SEQ ID NO: 5 (S-CO-AA-PP), SEQ IDNO: 6 (S-CO-AA-fneg-PP), and SEQ ID NO: 7 (S-CO-AA-fneg).

“Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

In an embodiment, the nucleic acid is recombinant. As used herein, theterm “recombinant” refers to (i) molecules that are constructed outsideliving cells by joining natural or synthetic nucleic acid segments tonucleic acid molecules that can replicate in a living cell, or (ii)molecules that result from the replication of those described in (i)above. For purposes herein, the replication can be in vitro replicationor in vivo replication.

The nucleic acid (e.g., DNA, RNA, cDNA, and the like) can be produced inany suitable matter including, but not limited to recombinant productionand commercial synthesis. In this respect, the nucleic acid sequence canbe synthetic, recombinant, isolated, and/or purified.

The nucleic acid can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Green et al. (eds.), Molecular Cloning, A Laboratory Manual,4^(th) Edition, Cold Spring Harbor Laboratory Press, New York (2012).For example, a nucleic acid can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides). Examples of modified nucleotides that can be used togenerate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid encoding the polypeptide can be provided as part of aconstruct comprising the nucleic acid and elements that enable deliveryof the nucleic acid to a cell, and/or expression of the nucleic acid ina cell. For example, the polynucleotide sequence encoding thepolypeptide can be operatively linked to expression control sequences.An expression control sequence operatively linked to a coding sequenceis ligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. Theexpression control sequences include, but are not limited to,appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG/AUG) in front of a protein-encoding gene, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons.

A large number of promoters, including constitutive, inducible, andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources of promoters include forexample, virus, mammal, insect, plant, yeast, and bacteria, and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available, for example, fromdepositories such as the ATCC as well as other commercial or individualsources. Promoters can be unidirectional (i.e., initiate transcriptionin one direction) or bi-directional (i.e., initiate transcription ineither a 3′ or 5′ direction). Non-limiting examples of promotersinclude, for example, the T7 bacterial expression system, pBAD (araA)bacterial expression system, the cytomegalovirus (CMV) promoter, theSV40 promoter, and the RSV promoter. Inducible promoters include, forexample, the Tet system, the Ecdysone inducible system, the T-REX™system (Invitrogen, Carlsbad, Calif.), LACSWITCH™ System (Stratagene,San Diego, Calif.), and the Cre-ERT tamoxifen inducible recombinasesystem.

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding region of the nucleic acid sequence and can mediatethe binding of regulatory factors, patterns of DNA methylation, orchanges in DNA structure. A large number of enhancers from a variety ofdifferent sources are well known in the art and are available as orwithin cloned polynucleotides (from, e.g., depositories such as the ATCCas well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMVpromoter) also comprise enhancer sequences. Enhancers can be locatedupstream, within, or downstream of coding sequences. For example, thenucleic acid encoding the polypeptide can be operably linked to a CMVenhancer/chicken (3-actin promoter (also referred to as a “CAGpromoter”).

A nucleic acid encoding the polypeptide can be cloned or amplified by invitro methods, such as the polymerase chain reaction (PCR), the ligasechain reaction (LCR), the transcription-based amplification system(TAS), the self-sustained sequence replication system (3SR) and the Q13replicase amplification system (QB). For example, a polynucleotideencoding the polypeptide can be isolated by polymerase chain reaction ofcDNA using primers based on the DNA sequence of the molecule. A widevariety of cloning and in vitro amplification methodologies are wellknown to persons skilled in the art.

An embodiment of the invention also provides a recombinant vectorcomprising the nucleic acid. Examples of suitable vectors includeplasmids (e.g., DNA plasmids), bacterial vectors, and viral vectors,such as poxvirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, poliovirus, alphavirus, baculovirus, measles virus, and Sindbisvirus. When the vector is a plasmid (e.g., DNA plasmid), the plasmid canbe complexed with chitosan.

The polypeptide, nucleic acid, or vector (e.g., recombinant measlesvirus vector) can be formulated as a composition (e.g., pharmaceuticalcomposition) comprising the polypeptide, nucleic acid, or vector (e.g.,recombinant measles virus vector) and a carrier (e.g., apharmaceutically or physiologically acceptable carrier). Furthermore,the polypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition can be used in the methods described hereinalone or as part of a pharmaceutical formulation.

The composition (e.g., pharmaceutical composition) can comprise morethan one polypeptide, nucleic acid, vector (e.g., recombinant measlesvirus vector), or composition of the invention. Alternatively, or inaddition, the composition can comprise one or more (e.g., one, two,three, or more) additional pharmaceutically active agents or drugs, suchas corticosteroids, antibiotics, and antivirals.

The carrier can be any of those conventionally used and is limited onlyby physio-chemical considerations, such as solubility and lack ofreactivity with the active compound(s) and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularpolypeptide, nucleic acid, vector, or composition thereof of theinvention and other active agents or drugs used, as well as by theparticular method used to administer the polypeptide, nucleic acid,vector (e.g., recombinant measles virus vector), or composition thereof.

The composition also can be formulated to enhance transductionefficiency. In addition, a person of ordinary skill in the art willappreciate that the one or more of the polypeptides, nucleic acids, orvectors (e.g., recombinant measles virus vectors) can be present in acomposition with other therapeutic or biologically-active agents. Forexample, factors that control inflammation, such as ibuprofen orsteroids, can be part of the composition to reduce swelling andinflammation associated with in vivo administration of one or more ofthe polypeptides, nucleic acids, or vectors (e.g., recombinant measlesvirus vector). Antibiotics, i.e., microbicides and fungicides, can bepresent to treat existing infection and/or reduce the risk of futureinfection.

The invention provides a method for preventing, inhibiting, reducing,eliminating, protecting, or delaying the onset of an infection or aninfectious clinical condition caused by coronavirus in a subjectcomprising administering the polypeptide, nucleic acid, vector (e.g.,recombinant measles virus vector), or composition thereof to a subject.The invention also provides a method for inducing an immune responseagainst a coronavirus in a subject comprising administering thepolypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof to the subject.

Administration of the polypeptide, nucleic acid, vector (e.g.,recombinant measles virus vector), or composition thereof to the subjectcan be used to protect against one or more strains of coronavirus (e.g.,SARS-CoV-2), thereby treating, preventing, and/or protecting againstcoronavirus-based pathologies.

Administration of the polypeptide, nucleic acid, vector (e.g.,recombinant measles virus vector), or composition thereof cansignificantly induce an immune response of a subject administered thepolypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof, thereby protecting against and treatingcoronavirus (e.g., SARS-CoV-2) infection.

Administration of the polypeptide, nucleic acid, vector (e.g.,recombinant measles virus vector), or composition thereof can induce ahumoral immune response in a subject. The induced humoral immuneresponse can be specific for the SARS-CoV-2 spike glycoprotein. Thehumoral immune response can be induced in the subject administered thevaccine by at least about 1.5-fold, at least about 2-fold, at leastabout 2.5-fold, at least about 3-fold, at least about 3.5-fold, at leastabout 4-fold, at least about 4.5-fold, at least about 5-fold, at leastabout 5.5-fold, at least about 6-fold, at least about 6.5-fold, at leastabout 7-fold, at least about 7.5-fold, at least about 8-fold, at leastabout 8.5-fold, at least about 9-fold, at least about 9.5-fold, at leastabout 10-fold, at least about 10.5-fold, at least about 11-fold, atleast about 11.5-fold, at least about 12-fold, at least about 12.5-fold,at least about 13-fold, at least about 13.5-fold, at least about14-fold, at least about 14.5-fold, at least about 15-fold, at leastabout 15.5-fold, at least about 16-fold, at least about 16.5-fold, atleast about 17-fold, at least about 17.5-fold, at least about 18-fold,at least about 18.5-fold, at least about 19-fold, at least about19.5-fold, at least about 20-fold, or ranges of any combination of thesevalues as compared to a subject not administered the polypeptide,nucleic acid, vector (e.g., recombinant measles virus vector), orcomposition thereof.

The induced humoral immune response can include an increased level ofneutralizing antibodies as compared to a subject not administered thepolypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof. The neutralizing antibodies can bespecific for the SARS-CoV-2 spike glycoprotein. The neutralizingantibodies can provide protection against and/or treatment of SARS-CoV-2infection and its associated pathologies in the subject administered thepolypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof.

The induced humoral immune response can include an increased level ofIgG antibodies as compared to a subject not administered thepolypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof. These IgG antibodies can be specificfor the SARS-CoV-2 antigens. The level of IgG antibody can be increasedby at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 4.5-fold, at least about 5-fold, at least about5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about7-fold, at least about 7.5-fold, at least about 8-fold, at least about8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about10-fold, at least about 10.5-fold, at least about 11-fold, at leastabout 11.5-fold, at least about 12-fold, at least about 12.5-fold, atleast about 13-fold, at least about 13.5-fold, at least about 14-fold,at least about 14.5-fold, at least about 15-fold, at least about15.5-fold, at least about 16-fold, at least about 16.5-fold, at leastabout 17-fold, at least about 17.5-fold, at least about 18-fold, atleast about 18.5-fold, at least about 19-fold, at least about 19.5-fold,at least about 20-fold, or ranges of any combination of these values ascompared to a subject not administered the polypeptide, nucleic acid,vector (e.g., recombinant measles virus vector), or composition thereof.

The induced humoral immune response can include an increased level ofIgM and/or IgA antibodies as compared to a subject not administered thepolypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof. These IgM and or IgA antibodies can bespecific for the SARS-CoV-2 antigen. The level of IgM/IgA antibody canbe increased by at least about 1.5-fold, at least about 2-fold, at leastabout 2.5-fold, at least about 3-fold, at least about 3.5-fold, at leastabout 4-fold, at least about 4.5-fold, at least about 5-fold, at leastabout 5.5-fold, at least about 6-fold, at least about 6.5-fold, at leastabout 7-fold, at least about 7.5-fold, at least about 8-fold, at leastabout 8.5-fold, at least about 9-fold, at least about 9.5-fold, at leastabout 10-fold, at least about 10.5-fold, at least about 11-fold, atleast about 11.5-fold, at least about 12-fold, at least about 12.5-fold,at least about 13-fold, at least about 13.5-fold, at least about14-fold, at least about 14.5-fold, at least about 15-fold, at leastabout 15.5-fold, at least about 16-fold, at least about 16.5-fold, atleast about 17-fold, at least about 17.5-fold, at least about 18-fold,at least about 18.5-fold, at least about 19-fold, at least about19.5-fold, at least about 20-fold, or ranges of any combination of thesevalues as compared to a subject not administered the polypeptide,nucleic acid, vector (e.g., recombinant measles virus vector), orcomposition thereof.

Administration of the polypeptide, nucleic acid, vector (e.g.,recombinant measles virus vector), or composition thereof can induce acellular immune response in the subject. The induced cellular immuneresponse can be specific for the SARS-CoV-2 antigen.

The induced cellular immune response can include eliciting a CD8+ T cellresponse, which can include eliciting a CD8+ T cell response in whichthe CD8+ T cells produce interferon-gamma (IFN-γ), tumor necrosis factoralpha (TNF-α), interleukin-2 (IL-2), or a combination thereof (e.g., acombination of IFN-γ and TNF-α).

The CD8+ T cell response can be increased by at least about 1.5-fold, atleast about 2-fold, at least about 2.5-fold, at least about 3-fold, atleast about 3.5-fold, at least about 4-fold, at least about 4.5-fold, atleast about 5-fold, at least about 5.5-fold, at least about 6-fold, atleast about 6.5-fold, at least about 7-fold, at least about 7.5-fold, atleast about 8-fold, at least about 8.5-fold, at least about 9-fold, atleast about 9.5-fold, at least about 10-fold, at least about 10.5-fold,at least about 11-fold, at least about 11.5-fold, at least about12-fold, at least about 12.5-fold, at least about 13-fold, at leastabout 13.5-fold, at least about 14-fold, at least about 14.5-fold, atleast about 15-fold, at least about 15.5-fold, at least about 16-fold,at least about 17-fold, at least about 18-fold, at least about 19-fold,at least about 20-fold, at least about 21-fold, at least about 22-fold,at least about 23-fold, at least about 24-fold, at least about 25-fold,at least about 26-fold, at least about 27-fold, at least about 28-fold,at least about 29-fold, at least about 30-fold, or ranges of anycombination of these values as compared to the subject not administeredthe polypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof.

Administration of the polypeptide, nucleic acid, vector (e.g.,recombinant measles virus vector), or composition thereof can alsoinclude eliciting a CD4+ T cell response, which can include eliciting aCD4+ T cell response in which the CD4+ T cells produce IFN-γ, TNF-α,IL-2, or a combination thereof (e.g., a combination of IFN-γ and TNF-α).

The polypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof can be administered to the subject byvarious routes including, but not limited to, oral, sublingual, buccal,intradermal, topical, parenteral (using single or arrays of dissolvableand hybrid microneedles, in lyophilized or solution), subcutaneous,intravenous, intramuscular, intraarterial, intrathecal, interperitoneal,intranasal, large and/or small particle aerosol, dry-powder aerosols orintratracheal administration, or subretinal injection or intravitrealinjection.

The invention includes a prime and boost protocol. In particular, theprotocol includes an initial “prime” with the polypeptide, nucleic acid,vector (e.g., recombinant measles virus vector), or composition thereof,followed by one or preferably multiple “boosts” with the polypeptide,nucleic acid, vector (e.g., recombinant measles virus vector), orcomposition thereof. The boosts can be administered 1-3 times (e.g., 1,2, or 3 times) at any suitable time period (e.g., every 3-4 weeks, everysix months, or once a year) for any suitable length of time.

The polypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof can be formulated in accordance withstandard techniques well known to those skilled in the pharmaceuticalart. Such compositions can be administered in dosages and by techniqueswell known to those skilled in the medical arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular subject, and the route of administration. The subject can bea mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog,a rat, or a mouse.

The polypeptide, nucleic acid, vector (e.g., recombinant measles virusvector), or composition thereof can be administered prophylactically ortherapeutically. In prophylactic administration, the polypeptide,nucleic acid, vector (e.g., recombinant measles virus vector), orcomposition thereof are administered in an amount sufficient to inducean immune response. In therapeutic applications, the polypeptide,nucleic acid, vector (e.g., recombinant measles virus vector), orcomposition thereof are administered to a subject in need thereof in anamount sufficient to elicit a therapeutic effect. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” Amountseffective for this use will depend on, e.g., the particular compositionof the polypeptide, nucleic acid, vector (e.g., recombinant measlesvirus vector), or composition thereof administered to a subject, themanner of administration, the stage and severity of the infection, thegeneral state of health of the patient, and the judgment of theprescribing physician.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example provides materials and methods for the studies described inExample 2.

Cell Lines, Viruses and Plasmids

Vero cells (African green monkey kidney cells; ATCC CCL-81) purchasedfrom ATCC (Manassas, Va., USA) were grown in advanced Dulbecco'smodified Eagle medium (DMEM; Gibco) supplemented with 10% (V/V) fetalbovine serum (HI FBS heat inactivated, Gibco) and GlutaMAX-I (Gibco).Cells were grown and maintained at 37° C. and 5% (V/V) CO2, and testednegative for mycoplasma contamination prior to use in virus rescueexperiments. MV plasmids expressing N, P and L, pMV^(EZ)EGFP(3), as wellas rMV^(EZ)EGFP(3) virus have been described previously (Rennick et al.,J. Virol. (2015). doi:10.1128/jvi.02924-14).

Construction of MV^(EZ) cDNA Plasmids Expressing SARS-CoV-2 SpikeGlycoprotein

As described in Rennick et al., J. Virol. (2015).doi:10.1128/jvi.02924-14), three recombinant viruses were generated thatcontained the open reading frame encoding enhanced green fluorescentprotein (EGFP) within an additional transcriptional unit (ATU) atvarious positions within the genome. rMV^(EZ)EGFP(1), rMV^(EZ)EGFP(3),and rMV^(EZ)EGFP(6) contained the ATU upstream of the N gene, followingthe P gene, and following the H gene, respectively. The viruses werecompared in vitro by growth curves, which indicated that rMV^(EZ)EGFP(1)was over-attenuated. Intratracheal infection of cynomolgus macaques withthese recombinant viruses revealed differences in immunogenicity.rMV^(EZ)EGFP(1) and rMV^(EZ)EGFP(6) did not induce satisfactory serumantibody responses, whereas both in vitro and in vivo rMV^(EZ)EGFP(3)was functionally equivalent to the commercial MV^(EZ)-containingvaccine. Intramuscular vaccination of macaques with rMV^(EZ)EGFP(3)resulted in the identification of EGFP⁺ cells in the muscle at days 3,5, and 7 post-vaccination. Therefore, rMV^(EZ)EGFP(3) was selected forfurther experiments.

pMV^(EZ) versions expressing SARS-CoV-2 spike glycoprotein (Accessionnumber MN908947) were generated by replacing the open reading frame ofEGFP in pMV^(EZ)EGFP(3) (Rennick et al., J. Virol. (2015).doi:10.1128/jvi.02924-14).

Plasmid pMV^(EZ)EGFP(3) was linearized using restriction sites Asc I atgenome position 3439 and Aat II at genome position 4176. Theserestriction sites were originally designed into pMV^(EZ)EGFP(3) to alloweasy replacement of foreign genes in place of EGFP. SARS-CoV-2 spikewith mutations in the endoplasmic reticulum (ER) retention signalsequence as previously described (Case et al., bioRxiv (2020).doi:10.1101/2020.05.18.102038) were obtained in two gene fragments.These fragments were ligated into the linearized pMV^(EZ)EGFP(3) usingGibson Assembly (NEBuilder® HiFi DNA assembly, NEB). This generated twoversions pMV^(EZ)SARS-CoV-2-S and pMV^(EZ)SARS-CoV-2-S6.

A human codon optimized SARS-CoV-2 spike glycoprotein expressing plasmidwas obtained from GenScript (Piscataway, N.J., USA). Spike was amplifiedfrom the plasmid using oligonucleotides that contained a 35 nucleotidehomology (lower case sequence) to the linearized pMV^(EZ)EGFP(3)(Forward primer:5′caaagtgattgcctcccaagttccacaggcgcgccATGTTCGTCTTCCTGGTC3′ (SEQ ID NO:24) and reverse primer:5′gttggcaggtaagttgagctgtaggacgtcgcgcgTTAGGTGTAATGCAGCTTCAC3′ (SEQ ID NO:25)). The amplified product was then ligated into linearizedpMV^(EZ)EGFP(3) using Gibson Assembly (NEBuilder® HiFi DNA assembly,NEB). This generated pMV^(EZ)SARSCoV2-S-CO. pMV^(EZ)SARS-CoV-2-S-COAAwas generated the same way, but using a reverse primer(5′ggttggcaggtaagttgagctgtaggacgtcgcgcgTTAGGTGTAAGCCAGCGCCACGCC3′ (SEQID NO: 26) with nucleotide changes (in bold) to disrupt the ER retentionsignal.

The spike sequence in all plasmids were sequence confirmed via Sangersequencing (Genewiz, N.J., USA).

Generation of Recombinant rMV^(EZ)-SARS-CoV-2-Spike Glycoprotein Viruses

Vero cell monolayers in 6-well trays were infected with a recombinantvaccinia virus expressing T7 polymerase (MVA-T7) in Opti-MEM (Gibco) for30 mins at 37° C. and then spinoculated at room temperature for another30 mins. Virus inoculum was removed and 1 ml fresh Opti-MEM was addedonto cells. Cells were then transfected with 5 μg ofpMV^(EZ)SARS-CoV-2-S, pMV^(EZ)SARS-CoV-2-S6, pMV^(EZ)SARS-CoV-2-S-CO, orpMV^(EZ)SARS-CoV-2-S-COAA. MV plasmids expressing nucleoprotein (N),phosphoprotein (P) and polymerase (L) at 1 μg, 0.6 μg and 0.4 μgrespectively, were also transfected into the cells. 24 hourspost-transfection (h.p.t.), medium was removed from the cells andDMEM/2% (V/V) fetal bovine serum (FBS) was added. Cells were monitoreddaily for approximately 14 days post-transfection (d.p.t.) for syncytiumformation. Plaque picked viruses were then grown in Vero cells andharvested by free-thaw when complete cytopathic effect was visible.Virus titers were determined by endpoint titration and expressed as 50%tissue culture infectious (TCID₅₀) units.

Reverse Transcription Polymerase Chain Reaction (RT/PCR) and Sequencing

Total RNA from working virus stocks was extracted using TRIzol LSreagent (ThermoFisher) according to manufacturer's recommendations andRNA pellet resuspended in 40 μl nuclease-free water (Invitrogen). cDNAwas generated with 5 μl of resuspended RNA using SuperScript™ IIIFirst-strand synthesis system (Thermo Fisher Scientific) and randomprimers. 3 μl of the resultant cDNA was then used to amplify MV-spikefragments with primers using Phusion high-fidelity DNA polymerase (NEB)in a total volume of 50 μl (using a touch-down PCR amplificationprotocol). Amplified PCR products were analyzed on a 1% agarose gel andbands gel purified using QIAquick gel extraction kit (Qiagen). Productswere sequenced confirmed via Sanger sequencing (Genewiz, N.J., USA).

Immunofluorescence

Confluent Vero cells in 24-well trays were infected withrMV^(EZ)SARS-CoV2-S6 or rMV^(EZ)SARS-CoV-2-S-CO at a multiplicity ofinfection (MOI) of 0.01. At 2 days post infection, cells were fixed with4% paraformaldehyde for 10 minutes at room temperature. Cells werewashed twice in PBS and permeabilized (0.1% Triton-X in PBS) at 37° C.for 30 minutes before incubating with primary antibody (Rabbitanti-SARS2-S, Sino biologicals 40150-R007; 1:500) made in PBS with 0.1%(V/V) Triton-X at 37° C. for 1 hour. Cells were then washed three timesin PBS before incubating with secondary antibody (Chicken anti-rabbitAlexa Fluor 488, Invitrogen; 1:400) at 37° C. for 1 hour. Cells werewashed three times in PBS and stained with DAPI nuclei stain(Invitrogen; 300 nM DAPI stain solution) for 10 minutes at roomtemperature in the dark. Images were obtained using a fluorescentmicroscope (Leica).

Virus Growth Kinetics

Vero cell monolayers at 2×10⁵ cells in 24-well trays were infected withrMV^(EZ)EGFP(3), rMV^(EZ)SARS-CoV-2-S3, rMV^(EZ)SARS-CoV-2-S6,rMV^(EZ)SARS-CoV-2-S-CO, or rMV^(EZ)SARS-CoV-2-S-COAA at a 0.05 MOI.Cell monolayers were infected for 1 hour at 37° C. after which virusinoculum was removed and cell monolayers washed twice usingphosphate-buffered saline (PBS; Gibco). DMEM/2% (V/V) FBS was added ontothe cells and at the desired time points cells were scraped into culturemedium and placed at −80° C. After freeze-thawing the cells and medium,cell debris were clarified and total virus measured by TCID₅₀.

Immunoplaque Assay

Confluent Vero cell monolayers in 6-well trays were infected with a10-fold serial dilution of virus prepared in Opti-MEM. Cells wereincubated for 1 h at 37° C. and then overlaid with 0.6% Avicel (FMCBiopolymer) supplemented with 2×MEM (10×MEM, no glutamine, Gibco) and 2%FBS. Cells were incubated for 5 days and then fixed using 4%paraformaldehyde for 30 minutes at room temperature. Cells werepermeabilized (0.5% Triton X-100, 20 mM sucrose in PBS; 1 ml) for 30minutes at room temperature and then washed (0.1% Tween-20 in PBS; 1 ml)once before incubating with primary antibody (Rabbit anti-SARS2-S, Sinobiologicals 40150-R007; 1:1000) in blocking buffer (4% dried milk/0.1%Tween-20 in PBS) for 1 hour at room temperature. Cells were washed threetimes and incubated with secondary antibody (Goat anti-rabbit HRP,Abcam, ab6721; 1:1000) for 1 hour at room temperature. Plaques werevisualized using KPL TrueBlue Peroxidase Substrate solution (Sera Care,5510-0050). Plates were digitalized using a scanner.

Animal Study Design

All animal experiments were conducted in compliance with all applicableU.S. Federal policies and regulations and AAALAC International standardsfor the humane care and use of animals. All protocols were approved bythe University of Pittsburgh Institutional Animal Care and Use Committee(IACUC).

Twenty-five IFNar1 knockout mice in groups of five were infected with10⁴ TCID₅₀ of rMV^(EZ)EGFP(3), rMV^(EZ)SARS-CoV-2-S3,rMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S-CO, orrMV^(EZ)SARS-CoV-2-S-COAA viruses via the intraperitoneal (IP) route. 21days post-infection, mice were bled for serum, and then boosted with 10⁵TCID₅₀ of the respective virus.

African green monkeys were immunized with either measles vaccine orrMV^(EZ)SARS-CoV-2-S-CO (in groups of 2 or 3) with 10⁵ TCID₅₀ ofcandidate vaccine. In our proof of principle study we focused on aprime/boost (day 0 and 21 days). Animals were challenged 42 days afterimmunization with 10⁶ plaque forming units of SARS-CoV-2.

All animals seroconverted generating antibodies to both measles andSARS-CoV-2. These antibodies neutralized both viruses, some at levelshigher than what is seen in human convalescent serum.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism software (LaJolla, Calif.).

Vector Maps

Vector maps are provided in FIGS. 4-10 for each ofrMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S-CO,rMV^(EZ)SARS-CoV-2-S-COAA, rMV^(EZ)SARS-CoV-2-S,rMV^(EZ)SARS-CoV-2-S-COAA-PP, rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP, andrMV^(EZ)SARS-CoV-2-S-COAA-fneg, wherein the spike glycoprotein isinserted after the measles virus N and P genes (in the third (3)position).

Example 2

This example demonstrates the generation and characterization ofrecombinant MV vaccine strain viruses expressing SARS-CoV-2 spikeglycoprotein.

Based on previously generated reverse genetics system forlive-attenuated measles virus (MV) vaccine strain Edmonston-Zagreb (EZ)that expresses an enhanced green fluorescent protein (EGFP; rMV^(EZ)EGFP(3)), recombinant MV vaccine viruses encoding SARS-CoV-2 spikeglycoprotein were generated and rescued. The open reading frame for thespike glycoprotein lies within an additional transcriptional unit atposition 3 in the MV genome in place of EGFP. These viruses werecompared in vitro for replication and expression of the spikeglycoprotein. The growth kinetics of all tested viruses were equivalentto rMV^(EZ) EGFP(3).

pMV^(EZ)EGFP(3) (Rennick et al., J. Virol. (2015).doi:10.1128/jvi.02924-14) was modified to express SARS-CoV-2 spikeglycoprotein in place of EGFP. In a manner similar to that described inCase et al, bioRxiv (2020). doi:10.1101/2020.05.18.102038, anendoplasmic reticulum (ER) retention signal sequence present in thecytoplasmic tail (CT) of the spike was altered from KxHxx to AxAxx-COOH,and anti-genomic plasmids expressing a non-codon and human codonoptimized version (pMV^(EZ)SARS-CoV2-S and pMV^(EZ)SARS-CoV-2-COAA,respectively) were generated. An additional human codon optimizedversion with the authentic KxHxx-COOH sequence was also generated(pMV^(EZ)SARS-CoV-2-CO).

Recovery of a plasmid with the correct spike sequence proved challengingfor the non-codon optimized version, and as a result two plasmids weregenerated: one with the authentic sequence (pMV^(EZ)SARS-CoV-2-S) andone with three nucleotide changes, one of which does not cause an aminoacid change (pMV^(EZ)SARS-CoV-2-S6).

To recover recombinant viruses, Vero cells were transfected with eitherpMV^(EZ)SARS-CoV-2-S, pMV^(EZ)SARS-CoV-2-S6, pMV^(EZ)SARS-CoV-2-00 orpMV^(EZ)SARS-CoV-2-COAA along with expression plasmids for nucleoprotein(N), phosphoprotein (P) and polymerase (L) proteins. Sequenceconfirmation of the virus generated from clone pMV^(EZ)SARS-CoV-2-Srevealed an amino acid change in the CT tail. This virus wassubsequently named rMV^(EZ)SARS-CoV-2-S3 (FIG. 1A).

The spike glycoprotein sequences in rMV^(EZ)SARS-CoV-2-S6,rMV^(EZ)SARS-CoV-2-CO, and rMV^(EZ)SARS-CoV-2-COAA correspond to theircDNA clones (FIG. 1A). Rescue experiments were reproducible withsyncytium formation about 14 days post transfection (FIG. 1B).

To test if the spike glycoprotein was expressed, Vero cells wereinfected and fixed and stained at 48 hours post-infection using ananti-SARS2-S antibody. Substantial amounts of spike expression weredetectable in the infected cells, especially for rMV^(EZ)SARS-CoV-2-CO(FIG. 1C). Expression was further confirmed by staining viral plaqueswith anti-SARS2-S antibody (FIG. 1D).

Growth analysis of rMV^(EZ)SARS-CoV-2-S3, rMV^(EZ)SARS-CoV-2-S6,rMV^(EZ)SARS-CoV-2-CO, and rMV^(EZ)SARS-CoV-2-COAA were compared torMV^(EZ)EGFP(3) in Vero cells over a 3 day period. Viruses replicatedsimilar to rMV^(EZ)EGFP(3) by reaching a titers close to 10⁷ TCID₅₀/ml(FIG. 2).

Additional vectors were generated with modifications to ablate the furincleavage signal (rMV^(EZ)SARS-CoV-2-S-COAA-fneg andrMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP), and to lock in the prefusionconformation by substitution of two proline residues(rMV^(EZ)SARS-CoV-2-S-COAA-PP and S-CO-AA-fneg-PP).

rMV^(EZ)SARS-CoV-2-S3 and rMV^(EZ)SARS-CoV-2-S6 encode a spikeglycoprotein with nucleotide changes that arose during virus rescue andplasmid generation, respectively.

rMV^(EZ)SARS-CoV-2-S-CO, rMV^(EZ)SARS-CoV-2-S-COAA,rMV^(EZ)SARS-CoV-2-S-COAA-PP, rMV^(EZ)SARS-CoV-2-S-COAA-fneg, andrMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP encode a human codon optimized spikeglycoprotein.

rMV^(EZ)SARS-CoV-2-S3, rMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S,rMV^(EZ)SARS-CoV-2-S-COAA, rMV^(EZ)SARS-CoV-2-S-COAA-PP,rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP, and rMV^(EZ)SARS-CoV-2-S-COAA-fnegcontain mutations in the spike glycoprotein to disrupt the endoplasmicreticulum (ER) retention sequence that has been described as essentialfor SARS-CoV-2 virion assembly via the ER-Golgi compartment.

The nucleic acid sequence encoding the spike glycoprotein ofrMV^(EZ)SARS-CoV-2-S3 corresponds to SEQ ID NO: 22.

The following table summarizes the sequence information for each of thevectors and the corresponding nucleic acid and amino acid sequences ofthe spike glycoprotein.

Nucleic Acid Amino Acid Sequence of Sequence of Nucleic Acid Spike SpikeSequence of Vector Designation Glycoprotein Glycoprotein VectorModifications rMV^(EZ)SARS-CoV-2-S6 1 8 15 modifications to ablate ERretention signal rMV^(EZ)SARS-CoV-2-S-CO 2 9 16 codon optimizedrMV^(EZ)SARS-CoV-2-S-COAA 3 10 17 codon optimized; modifications toablate ER retention signal rMV^(EZ)SARS-CoV-2-S 4 11 18 modifications toablate ER retention signal rMV^(EZ)SARS-CoV-2-S-COAA-PP 5 12 19 codonoptimized; modifications to ablate ER retention signal; modifications tolock in prefusion conformation rMV^(EZ)SARS-CoV-2-S-COAA-fneg-PP 6 13 20codon optimized; modifications to ablate ER retention signal;modifications to lock in prefusion conformation; modifications to ablatefurin cleavage signal rMV^(EZ)SARS-CoV-2-S-COAA-fneg 7 14 21 codonoptimized; modifications to ablate ER retention signal; modifications toablate furin cleavage signal rMV^(EZ)SARS-CoV-2-S3 22 modifications toablate ER retention signal

Example 3

This example describes vaccination/challenge studies with recombinant MVvaccine strain viruses expressing SARS-CoV-2 spike glycoprotein.

Mice

The experimental set-up for mice immunized with recombinant viruses isdescribed in FIG. 3A. Groups of 5 mice were infected with eitherrMV^(EZ)SARS-CoV-2-S3, rMV^(EZ)SARS-CoV-2-S6, rMV^(EZ)SARS-CoV-2-S-CO,rMV^(EZ)SARS-CoV-2-S-COAA, or rMV^(EZ)EGFP(3). Serum was collected ondays 21 and 42, and splenocytes were collected on day 42. Neutralizationof SARS-CoV-2 using the harvested mice serum indicates that SARS-CoV-2neutralizing antibodies were produced in mice vaccinated withrMV^(EZ)SARS-CoV-2-S-CO and rMV^(EZ)SARS-CoV-2-S-COAA viruses (FIG. 3B).

Splenocytes prepared from rMV^(EZ), rMV^(EZ)SARS-CoV-2-CO,rMV^(EZ)SARS-CoV-2-S6, and rMV^(EZ)SARS-CoV-2-COAA immunized mice wereused in an ELISPOT assay to detect the secretion of proinflammatorycytokine IFN-γ. Mice splenocytes were re-stimulated for 24 hours withfour pools of synthetic peptides (S1, S2, S3 and S4) designed to spanthe entire SARS-CoV-2 spike protein. Unstimulated splenocytes (medium)served as negative controls and splenocytes treated with PMA/ionomycinconfirmed splenocyte re-stimulation. The number of cells expressingIFN-γ after re-stimulation are represented as 1×10⁵ cells in FIG. 12.The results demonstrate that IFN-γ is secreted in spleens from miceimmunized with recombinant measles virus expressing SARS-CoV-2codon-optimized spike protein.

Flow cytometry was used to determine the proportion of CD4⁺/CD44⁺ andCD8⁺/CD44⁺ T cells in the spleens of mice immunized with rMV^(EZ),rMV^(EZ)SARS-CoV-2-CO, rMV^(EZ)SARS-CoV-2-S6, andrMV^(EZ)SARS-CoV-2-COAA. To determine the optimal re-stimulation periodsplenocytes from two mice from each immunized group were firstre-stimulated for 6 hours (FIG. 13A) while the remaining three micespleens were re-stimulated for 12-hours (FIG. 13B). Cells werere-stimulated with spike specific peptide pools S1, S2, S3 and S4, orleft unstimulated (negative control; medium). Re-stimulation wasconfirmed with a cell activation cocktail containing PMA/ionomycin andBrefaldin-A. Intracellular cytokine staining for IFN-γ and IL-13 werethen carried out to determine Th1 and Th2 responses, respectively. Theresults in FIGS. 13A and 13B demonstrate the T cell responses inimmunized mice.

Non-Human Primates

Multi-route delivery and challenge testing for natural measles isdescribed in de Swart et al., NPJ vaccines, 2(1), pp. 1-11 (2017).Specifically, infection of rhesus macaques with rMV^(EZ)EGFP(3) vaccinevia the intramuscular, intranasal, and aerosol routes, along with theintratracheal route as a control matching the usual experimentalinoculation route, has been described. Serum antibody responses weredetected by enzyme linked immunosorbent assay (ELISA), virusneutralization, or indirect immunofluorescence for MV fusion (MV-F) orhemagglutinin (MV-H) glycoprotein-specific antibodies. Animalsvaccinated by the intramuscular (IM) route produced similar antibodyresponses to those inoculated via the aerosol route. In all assays,lowest serum antibody levels were consistently observed in animalsimmunized by intranasal instillation. All animals were protected fromchallenge after intratracheal instillation of a wild-type strain of MV.

For this study, African green monkeys (AGMs) were used since aSARS-CoV-2 challenge model has been established (Hartman et al., doi:https://doi.org/10.1101/2020.06.20.137687 (2020)—in press PLoSPathogens). In this challenge model, AGMs were inoculated via amulti-route mucosal (oral, nasal, and ocular) exposure with a lowpassage, clinical isolate of SARS-CoV-2. The experimental design isdetailed in FIGS. 11A and 11B.

All AGMs develop mild disease with pulmonary lesions detectable byPET/CT in the acute phase which subsequently resolve. All AGMs exhibitprolonged shedding of infectious virus from oral, nasal, conjunctival,and rectal mucosal surfaces with viral RNA (vRNA) detectable throughoutthe respiratory and gastrointestinal tissues at later timepoints in theabsence of replication-competent virus.

Animals (maximum group size n=6) were vaccinated (IM) with therecombinant measles viral vector of the invention. Two animals werevaccinated with the standard measles virus Edmonston Zagreb vaccinestrain. Some animals received a boost at 3 or 4 weeks post-vaccination.Animals were sampled before vaccination and then weekly over 6 or 8weeks for the development of SARS-CoV-2 and MV serum antibody responses(IgG, IgM, IgA and neutralizing antibodies and T cell responses).

Development of MV-specific immune response will be compared tohistorical controls using banked samples from the studies analyzingintramuscular, aerosol, intranasal, and intratracheal administration ofMV vaccine (de Swart et al., NPJ vaccines, 2(1), pp. 1-11 (2017)). EDTAblood samples will be collected in Vacuette tubes containing K3EDTA asan anticoagulant. A sample of whole blood will be used directly forhematology analysis. Plasma will be separated from blood cells by lowspeed centrifugation and used in a commercial ELISA classic MeaslesVirus IgG assay (Serion) to assess anti-MV IgG alongside an in houseMV-N-specific IgG ELISA.

Development of anti SARS-CoV-2 specific IgG and IgM will be determinedusing an in house ELISA that detects antibodies against the SARS-CoV-2spike protein receptor binding domain (RBD). ELISA plates will be coatedwith 50 ng/well of SARS-CoV-2 RBD and subsequently blocked in 5% (V/V)FBS, 5% (WN) skim milk in PBS with 0.1% (V/V) Tween-20 (PBS T) for 1hour at 37° C. Serial dilutions of plasma will be made in block solutionand incubated on the blocked plates for 2 h at 37° C. After washing withPBS T, bound antibodies will be detected by incubation withgoat-anti-monkey IgM(p)-HRP (Seracare/KPL #5220-0334) orgoat-anti-rhesus IgG (H+L)-HRP (Southern Biotech #6200-05), both used ata 1:5,000 dilution in blocking solution for 1 hour at 37° C. Afterwashing with PBS T the assay will be developed by incubation with TMB(Seracare) for 7 min prior to the addition of TMB stop solution(Seracare). Absorbance values will be determined at 450 nm.

PBMC are isolated from the residual blood cell pellet by layeringdiluted whole blood onto Lymphoprep and subsequent density gradientcentrifugation. A sample of PBMC will be used for MV virus isolation byplating dilutions of PBMC with Vero cells expressing human CD150 (VerohCD150 cells). Assays are incubated at 37° C. for 3-5 days and thenscored for cytopathic effect. A portion of PBMC will also be used forRNA extraction and subsequent RT/PCR analysis for detection of viralgenome.

Blood samples for serum will be collected in Vacuette tubes (FIG. 11B).After coagulation, low speed centrifugation will be used to remove theclot from the serum supernatant. A sample will be used for serumbiochemistry and the rest will be available for neutralizing antibodyanalysis. Virus neutralizing antibodies will be detected using afluorescent focus reduction neutralization test (FRNT) for MV and aplaque reduction neutralization test (PRNT) for SARS-CoV-2. The FRNTuses a MV that expresses EGFP during replication; this facilitates rapidscreening of assays. Detection of fluorescence indicates virusreplication and seronegativity while lack of fluorescence indicates thepresence of neutralizing antibodies which prevent virus infection. Serumdilutions will be mixed with 100 plaque forming units (plu.) of MV andincubated at 37° C. for 1 hour. After addition of Vero cells expressingthe MV receptor human CD150, assays will be incubated at 37° C. for 3-5days before screening for fluorescence as a measure of virusreplication. For the PRNT, serum dilutions will be mixed with 100 plu.of SARS-CoV-2 and incubated at 37° C. for 1 hour after which they willbe added to confluent Vero E6 cell monolayers. After incubation at 37°C. for 1 hour, medium will be replaced by immunodiffusion agarose. Afterincubation at 37° C. for 72 hours, the agarose overlay will be removedand the cell monolayer fixed and stained with crystal violet. Plaqueswill be enumerated and the PRNT₈₀ calculated. The SARS-CoV-2 molecular,virological and immunological assays are described in Klimstra et al.,J. Gen. Virol., doi: 10.1099/jgv.0.001481 (2020).

The vaccinated and sham vaccinated animals will be challenged via multiroute mucosal exposure with 10⁶ plu. of SARS-CoV-2. Protection will beassessed by sampling oral, ocular, rectal and nasal swabs for decreasesin infectious virus and viral RNA copies following challenge, decreasedPET/CT signals in lungs and lymph nodes and secondary immune responses.Brushes will be used to collect throat and rectal samples and swabs willbe used to collect nasal and ocular samples into virus transport medium.After vortexing the brush/swab is removed and the remaining liquid isused directly for SARS-CoV-2 virus isolation by adding to VeroE6 cellmonolayers, and for RNA isolation for RTqPCR analysis. For virusisolations, after incubation at 37° C. for 1 hour, medium will bereplaced by immunodiffusion agarose. After incubation at 37° C. for 72hours, the agarose overlay will be removed and the cell monolayer fixedand stained with crystal violet to allow visualization of plaques. AfterRNA isolation, one-step RT-qPCR will be performed using a One-StepMultiplex RT-qPCR Supermix (BioRad), and primers and probe targeting theSARS-CoV-2 N gene sequence. Quantitation of virus genome copies will bedetermined by comparing the cycle threshold values from the unknownsamples to cycle threshold values from a positive-sense SARS-CoV-2 vRNAstandard curve generated from 10-fold serial dilutions of in housesynthesized template.

Serial PET/CT images will be acquired pre-infection and at 3 or 4 and 10or 11 dpi. The scans will be performed on a MultiScan LFER 150 (MedisoMedical Imaging Systems). CT acquisition will be performed using thefollowing parameters: Semi-circular single field-of-view, 360projections, 80 kVp, 670 μA, exposure time 90 ms, binning 1:4, voxelsize of final image: 500×500 μm. PET acquisition will be performed 55min after intravenous injection of 18F-fluoro-2-deoxy-D-glucose (FDG)with the following parameters: 10 min acquisition, single field-of-view,1-9 coincidence mode, 5 ns coincidence time window. PET images will bereconstructed with the following parameters: Tera-Tomo 3Dreconstruction, 400-600 keV energy window, 1-9 coincidence mode, medianfilter on, spike filter on, voxel size 0.7 mm, 8 iterations, 9 subsets,scatter correction on, attenuation correction based on CT material mapsegmentation. Images will be analyzed using OsiriX MD or 64-bit (v.11,Pixmeo, Geneva, Switzerland). Before analysis, PET images will beGaussian smoothed in OsiriX and smoothing will be applied to raw datawith a 3×3 matrix size and a matrix normalization value of 24. Wholelung FDG uptake will be measured by first creating a whole lungregion-of-interest (ROI) on the lung in the CT scan by creating a 3Dgrowing region highlighting every voxel in the lungs between −1024 and−500 Hounsfield units. This whole lung ROI will be copied and pasted tothe PET scan and gaps within the ROI will be filled in using a closingROI brush tool with a structuring element radius of 3. All voxels withinthe lung ROI with a standard uptake value (SUV) below 1.5 will be set tozero and the SUVs of the remaining voxels will be summed for a totallung FDG uptake (total inflammation) value. Thoracic lymph nodes will beanalyzed by measuring the maximum SUV within each lymph node using anoval drawing tool. Both total FDG uptake and lymph node uptake valueswill be normalized to back muscle FDG uptake measured by drawingcylinder ROIs on the back muscles adjacent to the spine at the sameaxial level as the carina (SUVCMR; cylinder-muscle-ratio). PETquantification values will be organized in Microsoft Excel and graphedusing GraphPad Prism.

Blood samples will be collected and processed as for the vaccinationphase of the study and the secondary immune response to SARS-CoV-2 willbe measured as before using the SARS-CoV-2 spike protein receptorbinding domain ELISA for detection of IgG and IgM, and the PRNT fordetection of neutralizing antibodies and the T cells assays alreadyestablished in the IFNAR mice (FIGS. 13A and 13B) to assess cellularimmune responses.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. (canceled)
 2. The method of claim 10, wherein the recombinant measlesviral vector is a recombinant Edmonston-Zagreb (EZ) measles viralvector.
 3. The method of claim 10, wherein the nucleic acid sequence hasbeen codon optimized.
 4. (canceled)
 5. The method of claim 10, whereinthe ER retention sequence of the SARS-CoV-2 spike glycoprotein containsAxAxx rather than KxHxx in the cytoplasmic tail.
 6. The method of claim10, wherein the nucleic acid sequence encoding the SARS-CoV-2 spikeglycoprotein is SEQ ID NO: 3 (S-CO-AA) .
 7. The method of claim 10,wherein the SARS-CoV-2 spike glycoprotein comprises the amino acidsequence of SEQ ID NO: 10 (S-CO-AA).
 8. The method of claim 10, whereinthe recombinant measles viral vector comprises the nucleic acid sequenceof SEQ ID NO: 17 (SARS-CoV-2-S-CO-AA).
 9. (canceled)
 10. A method forpreventing, inhibiting, reducing, eliminating, protecting, or delayingthe onset of an infection or an infectious clinical condition caused bycoronavirus in a subject comprising administering a recombinant measlesviral vector comprising a nucleic acid sequence encoding a Severe AcuteRespiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein tothe subject, wherein the nucleic acid sequence contains at least onemodification that disrupts the endoplasmic reticulum (ER) retentionsequence of the SARS-CoV-2 spike glycoprotein.
 11. A method for inducingan immune response against a coronavirus in a subject comprisingadministering a recombinant measles viral vector comprising a nucleicacid sequence encoding a SARS-CoV-2 spike glycoprotein to the subject,wherein the nucleic acid sequence contains at least one modificationthat disrupts the endoplasmic reticulum (ER) retention sequence of theSARS-CoV-2 spike glycoprotein.
 12. The method of claim 10, wherein thesubject is a human subject.
 13. The method of claim 10, wherein therecombinant measles viral vector is administered by oral, sublingual,buccal, intradermal, parenteral, subcutaneous, intravenous,intramuscular, intraarterial, intrathecal, or interperitonealadministration.
 14. A polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 10 (S-CO-AA). 15.-17.(canceled)
 18. A pharmaceutical composition comprising (i) thepolypeptide of claim 14, a nucleic acid encoding the polypeptide, or arecombinant vector comprising the nucleic acid and (ii) apharmaceutically acceptable carrier.
 19. A method for preventing,inhibiting, reducing, eliminating, protecting, or delaying the onset ofan infection or an infectious clinical condition caused by coronavirusin a subject comprising administering the polypeptide of claim 14, anucleic acid encoding the polypeptide, a recombinant vector comprisingthe nucleic acid, or a pharmaceutical composition thereof to thesubject.
 20. A method for inducing an immune response against acoronavirus in a subject comprising administering the polypeptide ofclaim 14, a nucleic acid encoding the polypeptide, a recombinant vectorcomprising the nucleic acid, or a pharmaceutical composition thereof tothe subject.
 21. The method of claim 11, wherein the recombinant measlesviral vector is a recombinant Edmonston-Zagreb (EZ) measles viralvector.
 22. The method of claim 11, wherein the nucleic acid sequencehas been codon optimized.
 23. The method of claim 11, wherein the ERretention sequence of the SARS-CoV-2 spike glycoprotein contains AxAxxrather than KxHxx in the cytoplasmic tail.
 24. The method of claim 11,wherein the nucleic acid sequence encoding the SARS-CoV-2 spikeglycoprotein is SEQ ID NO: 3 (S-CO-AA).
 25. The method of claim 11,wherein the SARS-CoV-2 spike glycoprotein comprises the amino acidsequence of SEQ ID NO: 10 (S-CO-AA).
 26. The method of claim 11, whereinthe recombinant measles viral vector comprises the nucleic acid sequenceof SEQ ID NO: 17 (SARS-CoV-2-S-CO-AA).